Mobile phase incompatibility

A well-known method for peak tracking is based on the phase-system switching idea, " which was developed to solve problems of mobile phase incompatibility in LC/MS target compound analysis. An analytical column is usually connected to a trapping column in tandem mode. A switching valve is placed after the UV detector, and the flow of nonvolatile eluents is directed through the trapping column to waste. When the peak of interest elutes from the analytical column it is... 

Three major difficulties have been generally met in directly combining LC with MS. The first concerns the ionization of nonvolatile and/or thermolabile analytes. The second is related to the mobile-phase incompatibility as result of the frequent use of nonvolatile mobile-phase buffers and additives in LC. The third is due to the apparent flow rate incompatibility as expressed in the need to introduce a mobile phase eluting from the column at a flow rate of 1 ml/min into the high vacuum of the MS. 

The fraction of column effluent containing the analyte and internal standard can be either collected manually for subsequent reinjection onto the second (analytical) column (offline operation) or diverted directly onto the second column via a high-pressure switching valve (online operation). For manual collection, a drop-counter-fraction collecting system rather than a volume collection system has been recommended (117). The fraction is collected in a small tapered tube, and the solvent is carefully evaporated off under a stream of nitrogen. The residue is then dissolved in a small volume of a suitable solvent for the analytical separation. Because the sample is reconstituted in offline operation, the potential problem of mobile phase incompatibility between the two HPLC systems is avoided, and hence any semipreparative/analytical combination can be used. 

The most appropriate way to solve mobile-phase incompatibility problems in LC-MS is to change the mobile-phase composition to LC-MS compatible conditions. This is the approach taken in most cases. Unfortrmately, it is not always possible to do so. In some cases, specialized LC colunrn materials demand a particular mobile-phase composition. This is the case with for instance some chiral colunms, which will only provide adequate enantiomeric separation in a predefined mobile phase. The retention behaviour in high-performance anion-exchange chromatography (HPAEC) is significantly influenced by the cation (sodium or ammonium) in the mobile phase. In these cases, mobile-phase... 

O-ring seals are less common in HPLC columns, but are quite feanUe. The main issue is the compatibility of the O-ring material with the mobile phase. Incompatible O-rings can disintegrate resulting in a sudden loss of the seal O-rings made of fluorocarbon-based rubbers such as Kalrez or Chemraz have a suffidently broad solvent compatibility to be useful with all molnle phases commonly used in HPLC. 

In SEC dimensions, the transfer of mixed mobile phases can affect molar mass calibration. In order to obtain reliable molar mass results, the calibration curves have to be measured using the extremes of mobile phase composition and tested for changes in elution behavior and pore-size influence in the SEC column packing. The better the thermodynamic property of the SEC eluent, the less influence is expected on the SEC calibration when the transfer of mobile phase from the previous dimension occurs. It has been shown to be advantageous to use the SEC eluent as one component of the mobile phase in the previous dimension to avoid potential interference and mobile phase incompatibility. 

The on-line combination of LC-LC-MS has been investigated for a number of reasons. In addition to the general prospects of multidimensional separation techniques, especially enhanced selectivity, there was special interest in the ability to perform LC-LC with two different mobile-phase compositions. In this way, it should be possible to avoid problems with mobile-phase incompatibility due to the use of nonvolatile mobile-phase constituents. A good example of this approach is the determination of enantiomers of p-blockers in plasma samples, described by Edholm and co-workers. Racemic mixtures of a p-blocker like metoprolol can be separated on a ai-acid glycoprotein column. However, the chromatography requires the use of a 20 mM phosphate buffer (pH 7) in the mobile phase, which is not compatible with on-line LC-MS. Therefore, the chiral column was coupled via a set of two trapping columns to a common reversed-phase LC column. After separation, the two enantiomers were sepa-... 

Multidimensional HPLC offers very high separation power when compared to monodimensional LC analysis. Thus, it can be applied to the analysis of very complex mixtures. Applications of on-line MD-HPLC have been developed, using various techniques such as heart-cut, on-column concentration or trace enrichment applications in which liquid phases on both columns are miscible and compatible are frequently reported, but the on-line coupling of columns with incompatible mobile phases have also been studied. 

This is not the case when high performance liquid chromatography (HPLC) and MS are considered where, due to the incompatibilities of the two techniques, they cannot be linked directly and an interface must be used, with its prime purpose being the removal of the chromatographic mobile phase. Unfortunately, no single... 

There are two major incompatibilities between HPLC and MS. The first is that the HPLC mobile phase is a liquid, often containing a significant proportion of water, which is pumped through the stationary phase (column) at a flow rate of typically 1 mlmin while the mass spectrometer operates at a pressure of around 10 torr (1.333 22 x 10 " Pa). It is therefore not possible simply to pump the eluate from an HPLC column directly into the source of a mass... 

Before considering these in detail, it is necessary to revisit the inherent incompatibilities between mass spectrometry and liqnid chromatography. These are, as discussed previously, that HPLC utilizes a liquid mobile phase, often containing significant amounts of water, flowing typically at 1 mlmin while the mass spectrometer must be maintained under conditions of high vacuum, i.e. around 10- torr (1.333 22 x IQ- Pa). 

The need for a more definitive identification of HPLC eluates than that provided by retention times alone has been discussed previously, as have the incompatibilities between the operating characteristics of liquid chromatography and mass spectrometry. The combination of the two techniques was originally achieved by the physical isolation of fractions as they eluted from an HPLC column, followed by the removal of the mobile phase, usually by evaporation, and transfer of the analyte(s) into the mass spectrometer by using an appropriate probe. 

The flame ionization detector Is the most popular of the flame-based detectors. Apart from a reduction in sensitivity compared to expectations based on gas chromatographic response factors [138] and incompatibility with the high flow rates of conventional bore columns (4-5 mm I. 0.), the flame ionization detector is every bit as easy to use in SFC as it is in gas chromatography [148,149]. It shows virtually no response to carbon dioxide, nitrous oxide and sulfur hexafluoride mobile phases but is generally incompatible with other mobile phases and mixed mobile phases containing organic modifiers except for water and formic acid, other gas chromatographic detectors that have been used in SFC include the thermionic ionization detector (148,150], ... 

The solvent in which a sample is dissolved can play a very important role in HPLC analysis. Immiscibility, precipitation, decomposition, and system peaks are all problems potentially caused by a sample solvent incompatible with the analysis. Ideally, the mobile phase should be identical to the reaction solvent. The addition of an internal standard permits a kinetic analysis to be conducted. 

Gas chromatography is a most favourable case for interfacing to a mass spectrometer, as the mobile phases commonly used do not generally influence the spectra observed, and the sample, being in the vapour phase, is compatible with the widest range of mass-spectral ionisation techniques. The primary incompatibility in the case of GC-MS is the difference in operating pressure for the two hyphenated instruments. The column outlet in GC is typically at atmospheric pressure, while source pressures in the mass spectrometer range from 2 to... 

While the first two difficulties have been overcome there is no general solution available for the problem of incompatibility of mobile-phase composition. LC-MS systems are more complicated than GC-MS, as the eluted substances are mostly involatile, co-eluted with solvent, and frequently not efficiently ionised by El or Cl processes. Solutions to the problem are various, including surface ionisation (SIMS, FAB, FD, HSI,... 

The mobile phase in LC-MS may play several roles active carrier (to be removed prior to MS), transfer medium (for nonvolatile and/or thermally labile analytes from the liquid to the gas state), or essential constituent (analyte ionisation). As LC is often selected for the separation of involatile and thermally labile samples, ionisation methods different from those predominantly used in GC-MS are required. Only a few of the ionisation methods originally developed in MS, notably El and Cl, have found application in LC-MS, whereas other methods have been modified (e.g. FAB, PI) or remained incompatible (e.g. FD). Other ionisation methods (TSP, ESI, APCI, SSI) have even emerged in close relationship to LC-MS interfacing. With these methods, ion formation is achieved within the LC-MS interface, i.e. during the liquid- to gas-phase transition process. LC-MS ionisation processes involve either gas-phase ionisation (El), gas-phase chemical reactions (Cl, APCI) or ion evaporation (TSP, ESP, SSI). Van Baar [519] has reviewed ionisation methods (TSP, APCI, ESI and CF-FAB) in LC-MS. 

There are two general types of multidimensional chromatography separation schemes those in which the effluent from one column flows directly on to a second column at some time during the experiment, and those in which some type of trap exists between the two columns to decouple them (off-line mode). The purpose of a trap is often to allow collection of a fixed eluate volume to reconcentrate the analyte zone prior to the second separation step, or to allow a changeover from one solvent system to another. The use of offline multidimensional techniques (conventional sample cleanup) with incompatible mobile phases, is common in the literature, and replacing these procedures with automated on-line multidimensional separations will require continuous development efforts. 

Table 7.89 lists the main characteristics of MDHPLC (see also Table 7.86). In MDHPLC the mobile-phase polarity can be adjusted in order to obtain adequate resolution, and a wide range of selectivity differences can be employed when using the various available separation modes [906]. Some LC modes have incompatible mobile phases, e.g. normal-phase and ion-exchange separations. Potential problems arise with liquid-phase immiscibility precipitation of buffer salts and incompatibilities between the mobile phase from one column and the stationary phase of another (e.g. swelling of some polymeric stationary-phase supports by changes in solvents or deactivation of silica by small amounts of water).

The use of extraction cartridges in the separation of azines, discussed in the last Section, is an example of on-column concentration using off-line column switching. A chromatogram can be cut off-line by collecting the zones of interest at the detector outlet followed by reinjection of the collected fraction onto a secondary column. The mobile phases used with the two columns should be compatible, eg they should be miscible and the mobile phase used with the first column should not have too high an eluting power in the second column. If the mobile phases are incompatible it may be possible to evaporate the primary mobile phase and redissolve the sample in a suitable solvent. 

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